This invention relates to adipocytes and more particularly to producing adipocytes from human mesenchymal stem cells.
Adipose tissue provides an energy storage reserve for the body in the form of triglycerides and this tissue can release free fatty acids when caloric intake falls below metabolic needs. In response to increased dietary intake, the body will normally automatically increase energy expenditure through activity to maintain an energy balance. Energy can also be released as heat. Adipose tissue is intimately involved in the maintenance of body temperature through brown adipose tissue and energy storage through white adipose tissue. There are normal energy regulation pathways that balance dietary intake with metabolic activity largely mediated through the hypothalamus. It is now also apparent that the adipocyte plays an active role in this process and likely produces molecules that serve to feed back and effect regulation of triglyceride metabolism.
The two types of adipose tissue, brown and white, carry out very different roles in the body. White adipose is, designed to store excess caloric intake while brown adipose tissue uses a unique system to syphon off excess calories and use it to generate body heat. The heat is generated in the mitochondria of brown adipose where oxidation of substrate is utilized to create a hydrogen ion gradient that is then collapsed in a regulated fashion generating heat instead of ATP. It has been shown that transgenic animals that lack brown adipose maintain efficient metabolism, are obese and continue to overeat (Lowell et al, 1993). Other rodent studies have also shown a link between obesity, continued overeating and a sensitivity to cold, suggesting a connection to the sympathetic nervous system (Friedman and Leibel, 1992)
Imbalance in energy metabolism in the body leads to several diseased states, most notably obesity and obesity-induced diabetes and these can be described as dysfunctions of energy storage tissues. A mutation in mice that leads to obesity was identified in 1950 (Ingalls et al., 1950) and the gene was recently identified by positional cloning. The product of the ob gene is a 16,000 MW protein named leptin or OB protein. Leptin is produced only by adipocytes and is a hormone which regulates the hypothalamus. A mutation has been identified in the mouse gene that results in premature termination of MRNA translation such that no functional leptin protein is made (Zhang et al. 1994). The role of leptin in regulation of lipid metabolism is an area of intense research. Recent published investigations include studies of the upstream promoter elements found adjacent to the ob gene which have been shown to bind C/EBP (or CCAAT/enhancer binding protein) (Yeh et al, 1995 and Hwang et al., 1996). Having a model experimental system for in vitro adipogenesis of human cells would provide for discoveries in this area.
Recently it has been reported that leptin may serve as a hormone that regulates fertility and may be the link between appropriate body weight and reproductive physiology (Chehab et al. 1996). Both underweight and overweight women have difficulty in conceiving and this is likely associated with hormonal imbalance in the body of these individuals. The connection between body weight, fertility and the leptin produced by adipocytes has been suspected and now tested in mice. When obese mice, which normally do not produce offspring without transplanting the ovaries to surrogate females, were injected with leptin, their body weight fell dramatically and they gave birth to their own litters (Chehab et al, 1996).
A variety of cell types have been shown to produce lipid containing vesicles under specific culture conditions. For example, mouse 3T3-L1 cells derived from NIH 3T3, an immortalized mouse cell line, can be grown and cultured as a fibroblastic cell. However, after exposure to dexamethasone and methyl-isobutylxanthine, the cells undergo differentiation which results in the production of intracellular lipid-containing vacuoles (Spiegelman and Green, 1981). Rat marrow stromal cells have been shown to undergo both osteogenic and adipogenic differentiation when cultured with fetal calf serum and dexamethasone, but the predominating cell type varies depending on conditions (Beresford et al., 1992). Specifically, when the steroid analog dexamethasone was present throughout the time course of culture, osteogenesis was favored; but when dexamethasone was present only during secondary culture, the adipogenetic pathway predominated as evidenced by lineage specific markers and cytological observation. Mouse derived CH3 10T1/2 cells are a multipotential cell line that, when treated with 5-azacytidine, undergoes terminal differentiation into adipocytes, myocytes and chondrocytes. The 5-azacytidine causes inhibition of DNA methylation and thus causes the activation of a few genes responsible for commitment to these lineages (Konieczny and Emerson, 1984).
In accordance with one aspect of the present invention, there is provided a composition and method for inducing human mesenchymal stem cells to preferentially differentiate into the adipogenic lineage, i.e., to differentiate into adipocytes.
Applicant has found that mesenchymal stem cells (MSCs) and in particular human mesenchymal stem cells (hMSCs) can be directed to differentiate into adipocytes by treating the human mesenchymal stem cells with (i) a glucocorticoid and (ii) a compound which elevates intracellular cAMP levels by either upregulating cAMP production or by inhibiting degradation of cAMP; in particular a compound which inhibits compound(s) which degrade cAMP.
Accordingly, in one aspect, the human mesenchymal stem cells are treated with a glucocorticoid and a compound which inhibits the activity of a compound which degrades cAMP; in particular a phosphodiesterase inhibitor. The cells are subsequently cultured in media containing insulin and fetal bovine serum.
In a preferred aspect the human mesenchymal stem cells are treated with a glucocorticoid; insulin; and at least two compounds which inhibit degradation of cAMP, wherein one said compound which inhibits degradation of cAMP is indomethacin.
In a particularly preferred aspect, the human mesenchymal stem cells are treated with a glucocorticoid; a compound which inhibits the activity of a compound which degrades cAMP; insulin; and a compound which upregulates peroxisome proliferator activated receptor xcex3(PPAR xcex3) expression and/or increases its binding affinity to its DNA binding site.
In a further embodiment, the invention provides a composition comprising MSCs grown on a stabilized collagen gel matrix which are induced to differentiate into adipocytes.
Human mesenchymal stem cells, as well as their isolation and expansion, have been described in U.S. Pat. No. 5,486,359. As known in the art, human mesenchymal stem cells are capable of producing two or more different types (lineages) of mesenchymal cells or tissues and in particular connective tissue. The present invention provides a method for generating adipocytes from primary human mesenchymal stem cells in a predictable and reproducible manner. The invention is unique in that it involves human cells in primary and passaged cultures rather than transformed or immortalized cell lines that are predetermined to enter the adipogenic pathway. hMSCs are capable of entering multiple lineages including the osteocytic, chondrocytic, myocytic, tendonocytic and stromogenic lineages and the present invention provides a method and composition for inducing hMSCs to differentiate into adipocytes. In a preferred aspect, in accordance with the present invention, hMSC""s are induced to differentiate into essentially only adipocytes, i.e., there is no essential production or commitment to cells of other mesenchymal lineages. The method may also be used for generating adipocytes from MSCs from other species such as rabbit, dog, rat and mouse.
The invention also provides methods to purify the adipocytes to obtain a highly purified population.
The method of the invention for the in vitro differentiation of human mesenchymal stem cells preferably derived from bone marrow into adipoblastic or adipocytic cells is useful to investigators wishing to study this developmental program in human cells in vitro. A better understanding of diseases of energy metabolism including obesity and obesity-related diabetes will also result from studies of the differentiation of mesenchymal stem cells to adipocytes. While a cellular and biochemical basis for obesity has long been suspected, advancements have been slow due to a lack of model systems with biochemical and molecular tools for study. Recent dramatic breakthroughs in the molecular basis of adipogenesis have opened new avenues towards understanding this pathway of mesenchymal cell differentiation, although a human model system such as the one described here has been lacking. The method will also have utility in the isolation and preparation of adipocytes for implantation into a patient for the purpose of tissue augmentation following trauma or cosmetic surgery.